Transmembrane nature of chromaffin granule dopamine beta-monooxygenase.
The topographical arrangement of membrane-bound dopamine beta-monooxygenase in the chromaffin granule was investigated by proteolytic treatment and radiolabeling of intact granules or the isolated granule membranes. Topographical analysis was furthered by precipitating detergent extracts of treated membranes with specific antibodies to dopamine beta-monooxygenase, followed by electrophoresis of the immunoprecipitate in acrylamide-dodecyl sulfate. Trypsin or pronase treatment of intact granules cleaved small portions of membrane-bound dopamine beta-monooxygenase, resulting in proteins with slightly lower molecular weights. Membranes bearing these smaller forms of membrane-bound dopamine beta-monooxygenase were fully active enzymatically. Pronase treatment of isolated membranes cleaved all of the membrane-bound dopamine beta-monooxygenase antigenic sites from the membrane and also removed the enzymatic activity. Lactoperoxidase-catalyzed iodination revealed that membrane-bound dopamine beta-monooxygenase can be labeled in intact granules, but to a greater extent in isolated membranes. Quantitative peptide mapping showed that the distribution of label among tryptic peptides of membrane-bound dopamine beta-monooxygenase differed, depending on whether intact granules or isolated membranes had been labeled. The results of these experiments provide evidence that membrane-bound dopamine beta-monooxygenase is a transmembrane protein which has its active site exclusively on the intragranular face, plus a unique portion of its polypeptide chain exposed on the cytoplasmic face of the intact granule.
D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology*; D002838:Chromaffin System / Q000201:enzymology*; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism*; D005779:Immunodiffusion; D007425:Intracellular Membranes / Q000201:enzymology*; D011402:Pronase / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D004299:Dopamine beta-Hydroxylase; D014357:Trypsin; D011402:Pronase